Multi-omics and pathway analyses of genome-wide associations implicate regulation and immunity in verbal declarative memory performance

Background Uncovering the functional relevance underlying verbal declarative memory (VDM) genome-wide association study (GWAS) results may facilitate the development of interventions to reduce age-related memory decline and dementia. Methods We performed multi-omics and pathway enrichment analyses of paragraph (PAR-dr) and word list (WL-dr) delayed recall GWAS from 29,076 older non-demented individuals of European descent. We assessed the relationship between single-variant associations and expression quantitative trait loci (eQTLs) in 44 tissues and methylation quantitative trait loci (meQTLs) in the hippocampus. We determined the relationship between gene associations and transcript levels in 53 tissues, annotation as immune genes, and regulation by transcription factors (TFs) and microRNAs. To identify significant pathways, gene set enrichment was tested in each cohort and meta-analyzed across cohorts. Analyses of differential expression in brain tissues were conducted for pathway component genes. Results The single-variant associations of VDM showed significant linkage disequilibrium (LD) with eQTLs across all tissues and meQTLs within the hippocampus. Stronger WL-dr gene associations correlated with reduced expression in four brain tissues, including the hippocampus. More robust PAR-dr and/or WL-dr gene associations were intricately linked with immunity and were influenced by 31 TFs and 2 microRNAs. Six pathways, including type I diabetes, exhibited significant associations with both PAR-dr and WL-dr. These pathways included fifteen MHC genes intricately linked to VDM performance, showing diverse expression patterns based on cognitive status in brain tissues. Conclusions VDM genetic associations influence expression regulation via eQTLs and meQTLs. The involvement of TFs, microRNAs, MHC genes, and immune-related pathways contributes to VDM performance in older individuals. Supplementary Information The online version contains supplementary material available at 10.1186/s13195-023-01376-6.

American participants were excluded from this analysis to limit the potential for false positive associations due to population stratification.Among white participants, genotyping was attempted in 3,397 participants and was successful in 3,295 persons.As part of the CHS Cognition Study 11,12 , in 1992-94 and again, in 1997-99, participants were invited to undergo a detailed neuropsychological assessment including the CVLT 13 and the logical memory test from the WMS-R 14 .Among participants with genome-wide data: after exclusion of 47 participants with dementia and of 26 participants with history of stroke, 334 participants were available a GWAS of delayed word list recall (CVLT); after exclusion of 4 participants with dementia, 472 participants were available for a GWAS of delayed paragraph recall.

Framingham Heart Study (FHS)
The FHS is a three-generation, single-site, community-based, prospective cohort study that was initiated in 1948 to investigate risk factors for cardiovascular disease including stroke.It now comprises 3 generations of participants: the original cohort followed since 1948 (Original) 15 ; their offspring and spouses of the offspring, followed since 1971 (Offspring) 16 ; and children from the largest offspring families enrolled in 2000 (Gen 3) 17 .The Original cohort enrolled 5,209 men and women who comprised two-thirds of the adult population then residing in Framingham, MA, USA.Survivors continue to receive biennial examinations.The Offspring cohort comprises 5,124 persons (including 3,514 biological offspring) who have been examined approximately once every 4 years.Participants in the first two generations were invited to undergo an initial neuropsychological test battery in 1999-2005 18 , including the logical memory test from the Original WMS 19 .Neuropsychological testing in Gen 3 only began in 2009 and is not included in these analyses.The population of Framingham was virtually entirely whites in 1948 when the Original cohort was recruited.Vascular risk factors and outcomes, including transient ischemic attack, stroke and dementia, were identified prospectively since 1948 through an ongoing system of FHS clinic and local hospital surveillance 20,21 .Participants had DNA extracted and provided consent for genotyping in the 1990s.Genotyping was performed at Affymetrix (Santa Clara, CA) through an NHLBI funded SNP-Health Association Resource (SHARe) project and was successful in 4,519 persons from the Original and Offspring cohorts.Of these 4,519 persons, 4,116 were alive in 1999 when the neuropsychological study began.Of these, 2,642 participants have undergone neuropsychological testing including logical memory.We excluded 30 participants with a neurological condition that might confound the cognitive assessment (e.g.brain tumor or severe head injury), 7 participants with dementia and 52 participants with a history of stroke.Of the remaining 2,553 participants, 2,493 were aged 45 years or older and were available for a GWAS of delayed paragraph recall.

Lothian Birth Cohort 1921 (LBC1921) and 1936 (LBC1936)
These samples include surviving participants from the Scottish Mental Surveys of 1932 or 1947 (SMS1932 and SMS1947), having been born, respectively in 1921 (LBC1921) and 1936 (LBC1936) [22][23][24] .They were all Caucasian and almost all lived independently in the Lothian region (Edinburgh city and surrounding area) of Scotland.The LBC1921 cohort comprised 550 members while the LBC1936 cohort included 1,091 participants.At age 79 approximately, LBC1921 participants underwent a neuropsychological examination including the logical memory test from the WMS-R 14 .At age 70, LBC1936 participants took a battery of cognitive tests 22 , including the logical memory test from the Wechsler Memory Scale-IIIUK (WMS-IIIUK) 25 .
Genotyping was performed at the Wellcome Trust Clinical Research Facility (WTCRF) Genetics Core, Western General Hospital, Edinburgh.Among participants with good quality genome-wide data, after exclusion of 5 participants for dementia and 43 participants for history of stroke, 469 individuals from the LBC1921 cohort were available for a GWAS on paragraph delayed recall .Among participants with genome-wide data, after exclusion of 50 participants for history of stroke, 953 individuals from the LBC1936 cohort were available for a GWAS on delayed paragraph recall.

Rush Memory and Aging Project (MAP)
The MAP, started in 1997, enrolled older men and women from assisted living facilities in the Chicago area with no evidence on dementia at baseline 26 .Since October 1997, 1,285 participants completed their baseline evaluation, of whom 1,118 were non-Hispanic white.The study was approved by the institutional review board of Rush University Medical Center.The follow-up rate of survivors exceeds 90%.Similar to the ROS, participants agreed to annual clinical evaluations and signed both an informed consent and an Anatomic Gift Act form donating their brains, spinal cords, and selected nerves and muscles to Rush investigators at the time of death 27,28 .A more detailed description of the MAP has been published previously 27,28 .Participants were invited to take a neuropsychological test battery, including delayed recall of Story A from the logical memory subset of the Wechsler Memory Scale-Revised 14 , and delayed word list recall from the CERAD battery 29 .DNA was extracted from whole blood, lymphocytes, or frozen postmortem brain tissue.Genotyping was performed at the Broad Institute's Center for Genotyping and the Translational Genomics Research Institute.Among participants with available memory tests and genome-wide genotypes, after exclusion of 51 participants with dementia and 86 participants with a history of stroke, 751 individuals were available for a GWAS of delayed paragraph recall and delayed word list recall (visually presented word list).

Orkney Complex Disease Study (ORCADES)
ORCADES is an ongoing, family-based, cross-sectional study that seeks to identify genetic factors influencing cardiovascular and other disease risk in the population isolate of the Orkney Isles in northern Scotland 30 .The North Isles of Orkney, the focus of this study, consist of a subgroup of ten inhabited islands with census populations varying from ~30 to ~600 people on each island.The first phase of data collection was carried out in Orkney between 2005 and 2007.Informed consent and blood samples were provided by 1019 Orcadian volunteers who had at least one grandparent from the North Isles of Orkney.Participants were invited to take a neuropsychological test battery including the logical memory test adapted from the Original Wechsler Memory Scale 19 .Genotyping was performed at the Helmholtz Centre in Munich on a subset of 719 participants.An additional 169 individuals were genotyped by Integragen in Paris.Among participants with genome-wide data, we excluded 7 participants with a history of stroke.Of the 537 remaining individuals, aged 20 years or older, 419 participants aged 45 years or older were available for a GWAS of delayed paragraph recall.

Religious Orders Study (ROS)
The ROS, started in 1994, enrolled Catholic priests, nuns, and brothers, aged 53 years from about 40 groups in 12 states 26 .Since January 1994, 1,132 participants completed their baseline evaluation, of whom 1,001 were non-Hispanic white.The study was approved by the institutional review board of Rush University Medical Center.The follow-up rate of survivors exceeds 90%.Participants were free of known dementia at enrollment, agreed to annual clinical evaluations, and signed both an informed consent and an Anatomic Gift Act form donating their brains at time of death 27 .A more detailed description of the ROS has been published previously 27 .Participants were invited to take a neuropsychological test battery, including delayed recall of Story A from the logical memory subset of the Wechsler Memory Scale-Revised 14 , and delayed word list recall from the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) battery 29 .DNA was extracted from whole blood, lymphocytes, or frozen postmortem brain tissue.Genotyping was performed at the Broad Institute's Center for Genotyping and the Translational Genomics Research Institute.Among participants with available memory tests and genome-wide genotypes, after exclusion of 58 participants with dementia and 65 participants with a history of stroke, 687 individuals were available for a GWAS of delayed paragraph recall and delayed word list recall (visually presented word list).

Aging Gene-Environment Susceptibility -Reykjavik Study (AGES-Reykjavik)
The AGES-Reykjavik Study is a single center prospective cohort study based on the Reykjavik Study.The Reykjavik Study was initiated in 1967 by the Icelandic Heart Association to study cardiovascular disease and risk factors.The cohort included men and women born between 1907 and 1935 who lived in Reykjavik at the 1967 baseline examination.Re-examination of surviving members of the cohort was initiated in 2002 as part of the AGES-Reykjavik Study.The AGES-Reykjavik Study is designed to investigate aging using a multifaceted comprehensive approach that includes detailed measures of brain function and structure.All cohort members were European Caucasians.Briefly, as part of a comprehensive examination, all participants answered a questionnaire, underwent a clinical examination and had blood drawn 31 .All consenting participants were offered to take a neuropsychological test battery 32 , including the California Verbal Learning Test (CVLT) 13 , to assess memory performance.Among participants with genome-wide data, after exclusion of 126 participants with dementia and 266 participants with a history of stroke, 2616 participants were available for a GWAS of delayed word list recall (CVLT).

Erasmus Rucphen Family (ERF)
The Erasmus Rucphen Family (ERF) study is a family-based cohort study in a genetically isolated population in the Netherlands 33,34 , including 3,000 participants.Participants are all descendants of a limited number of founders living in the 19 th century.Extensive genealogical data is available for this population.The study protocol included venous puncture for DNA isolation and chemistry, cognitive evaluation, cardiovascular examination, eye assessments and body composition measurements.All participants gave informed consent and the study was approved by the medical ethics committee at Erasmus MC University Medical Center.Genotyping was done at the Human Genotyping Facility, Genetic Laboratory Department of Internal Medicine, Erasmus MC, Rotterdam, and at the Genotyping Center of Leiden University, The Netherlands.In total, 2,385 samples from the ERF Study were available with good quality genotyping data.Participants were invited to undergo a neuropsychological evaluation 35 , which included the Dutch version of Rey's Auditory Verbal Learning Test (RAVLT) 36 .Among participants with genome-wide data, we excluded 20 individuals with a history of stroke.Of the remaining 2 119 participants, aged 20 years or older, 1,267 participants were aged 45 years or more and were available for performance GWAS of delayed word list recall (RAVLT).

Genetic Epidemiology Network of Arteriopathy (GENOA)
GENOA is a study of hypertensive sibships designed to investigate the genetic underpinnings of hypertension and target organ damage.In the initial phase of the GENOA study (Phase I: 1996-2001), all members of sibships containing ≥ 2 individuals with essential hypertension clinically diagnosed before age 60 were invited to participate, including both hypertensive and normotensive siblings (1,583 non-Hispanic whites from Rochester, MN, and 1,841 African Americans from Jackson, MS).The diagnosis of essential hypertension was established based on blood pressure levels measured at the study visit (>140 mmHg average systolic BP or >90 mmHg average diastolic BP) or a prior diagnosis of hypertension and current treatment with antihypertensive medications.Exclusion criteria were secondary hypertension, alcoholism or drug abuse, pregnancy, insulin-dependent diabetes mellitus, or active malignancy.In the second phase of the GENOA study (Phase II: 2000-2004), 1,241 white and 1,482 African American participants were successfully re-recruited to measure potential target organ damage due to hypertension.The Genetics of Microangiopathic Brain Injury (GMBI) study (2001-2006) is an ancillary study of GENOA undertaken to investigate susceptibility genes for ischemic brain injury.Phase II GENOA participants that had a sibling willing and eligible to participate in the GMBI study underwent a neurocognitive testing battery to assess several domains of cognitive function including Rey's Auditory Verbal Learning Test (RAVLT) 36 (967 whites and 1,010 African Americans).Genotyping was performed at the Mayo Clinic, Rochester (MN).GENOA white participants who were less than 45 years of age (N=56) or had history of stroke (N=22) were excluded from the analysis, leaving a total of 889 participants.Among participants with genome-wide genotype data, 758 GENOA white participants from 378 sibships were available for a GWAS on delayed word list recall (RAVLT).

Helsinki Birth Cohort Study (HBCS)
The source cohort for the HBCS comprised 4,130 women and 4,630 men born as singletons at Helsinki University Central Hospital during 1934-44, who had birth and child welfare records and were living in Finland in 1971 37 .To approach an intended sample size of N=2,000, a random subsample of 2,902 subjects was invited to participate in the study; 2,003 of them (1,075 women and 928 men) were finally included 38 .Participants who could come to the examination center were invited to take a neuropsychological test battery, including the delayed word list recall from the CERAD battery 29 .1,063 participants attended neuropsychological testing between February 2005 and February 2011.DNA was extracted from 1,728 randomly selected participants of the HBCS.Genotyping was conducted at the Wellcome Trust Sanger Institute, Cambridge, UK.Among participants with available cognitive tests and genome-wide genotypes, after exclusion of 19 participants with a history of stroke, 888 individuals were available for a GWAS of delayed word list recall (visually presented word list).The study was approved by the Institutional Review Board of the National Public Health Institute, and informed consent was obtained from all participants.

Croatian Cohorts: Split and Korčula
The Croatia-Korčula study is part of a larger genetic epidemiology research program in Croatian island isolates, "10,001 Dalmatians."The genetic epidemiology research program in Croatian island isolates began in 1999 39 , then expanded to study human genetic variation and effects of isolation and inbreeding 40,41 , and finally entered the phase of focusing on diseases and gene mapping studies [42][43][44] .A total of 969 participants were included in the CROATIA-Korčula study.The Croatia-Split study included 535 persons collected in 2009 from the general (outbred) population of Split.Split has a population of >100,000 and is the second largest city in Croatia.Participants from the Croatia-Korčula and Croatia-Split studies were invited to undergo a neuropsychological examination including the Rey's Auditory Verbal Learning Test (RAVLT) 36 .Croatia-Korčula genotyping was performed at the Institute of Human Genetics, Helmholtz Zentrum München, Germany and CROATIA-Split genotyping was performed at AROS Applied Biotechnology, Aarhus, Denmark.Genotyping was successful in 898 and 499 participants, respectively, for Croatia-Korčula and Croatia-Split.Among participants with genome-wide data, 25 and 10 participants with a history of stroke were excluded from the Croatia-Korčula and Croatia-Split studies, respectively.Of the remaining 577 and 471 individuals, aged 20 years or more, 472 and 303 participants aged 45 years or older were available for a GWAS of delayed word list recall (RAVLT).

Rotterdam Study
The Rotterdam Study is a population-based prospective cohort study among inhabitants of a district of Rotterdam (Ommoord), The Netherlands, and aims to examine the determinants of disease and health in the elderly with a focus on neurogeriatric, cardiovascular, bone, and eye disease 45 46,47 .In 1990-1993, 7,983 persons 55 years of age or over (Rotterdam Study-I) participated and were re-examined every 3 to 4 years.In 1999, 3,011 individuals who had become 55 years of age or moved into the study district since the start of the study were added to the cohort (Rotterdam Study-II), and in 2006 a further extension of the cohort was initiated in which 3,932 subjects aged 45-54 years and living in the same district were included (Rotterdam Study-III) 48 .All participants had DNA extracted at their first visit.Genotyping was attempted in participants with high-quality extracted DNA.Genotyping was done at the Human Genotyping Facility, Genetic Laboratory Department of Internal Medicine, Erasmus MC, Rotterdam, the Netherlands.Participants underwent several neuropsychological tests at the baseline and follow-up examinations 49 , including a 15-word verbal learning test based on Rey's recall of words 50 .Participants are continuously monitored for major events, including dementia and stroke, by automated linkage of the general practitioners' records and hospital discharge files with the study database 51,52 .Among participants with good quality genome-wide data: after exclusion of participants with dementia (N=124 for the Rotterdam Study-I, N=56 for the Rotterdam Study-II and N=6 for the Rotterdam Study-III) and participants with a history of stroke (N=168 for the Rotterdam Study-I and N=102 for the Rotterdam Study-II), 2,067 participants from the Rotterdam Study-I, 1,533 participants from the Rotterdam Study-II and 1,935 participants from the Rotterdam Study-3 were available for a GWAS of word list delayed recall (visually presented word list).

Study of Health in Pomerania (SHIP)
The Study of Health in Pomerania is a population-based epidemiological study in the region of Western Pomerania, Germany 53 .In brief, from the total population of West Pomerania comprising 213,057 inhabitants in 1996, a two-stage stratified cluster sample of adults aged 20-79 years was drawn.The net sample (without migrated or deceased persons) comprised 6,265 eligible subjects, out of which 4,308 completed their baseline examinations.From July 2007 to October 2010 the Life-Events and Gene-Environment Interaction in Depression (LEGENDE) study was carried out in the SHIP cohort.A diagnostic interview for mental disorders was performed based on Diagnostic and Statistical Manual for Mental Disorders (IV edition) diagnostic criteria 54 .As part of the SHIP-LEGENDE project 55 , participants have been invited to take a cognitive test battery, including a German adaptation of Rey's Auditory Verbal Learning Test (RAVLT).Genotyping was performed at Affymetrix (Santa Clara, CA).The genetic data analysis workflow was created using the Software InforSense.Genetic data were stored using the database Caché (InterSystems).Among participants with available cognitive tests and genome-wide genotypes, we excluded 62 participants with dementia and 19 participants with a history of stroke.Of the remaining 2,027 individuals, aged 20 years or older, 1,474 participants aged 45 years or older were available for a GWAS of delayed word list recall (RAVLT).

Tasmanian Study of Cognition and Gait (TASCOG)
TASCOG is a study of cerebrovascular mechanisms underlying gait, balance and cognition in a populationbased sample of Tasmanian people aged at least 60 years.Individuals aged 60-86 years (N=395) living in Southern Tasmania, Australia, were randomly selected from the electoral roll to participate in the study.Individuals were excluded if they lived in a nursing home, had a contraindication for magnetic resonance scanning (MRI) or were unable to walk without a gait aid 56 .Participants were invited to take a neuropsychological test battery 57 , including among other tests the Hopkins Verbal Learning Test (HVLT) 58 .DNA was extracted from peripheral blood samples by proteinase K digestion following cell lysis, then phenolchloroform purification.DNA was genotyped at the Diamantina Institute and Institute of Molecular Biosciences, University of Queensland, Australia, for 370 participants, and call rates were greater than 97% for all samples.Genotypes for 22 individuals were excluded, either because they were closely related to other individuals, they were outliers in a population ancestry analysis or their sex predicted from genotypes did not match sex as recorded in the database.Among the 348 remaining participants with available genome-wide data, after exclusion of 2 participants with dementia and 15 participants with a history of stroke, 331 individuals were available for performance GWAS of delayed word list recall (HVLT).

Replication Studies with Memory GWAS
Three cohorts in the replication study of the GWAS analysis 2 have also completed the whole-genome association analysis and been investigated as replication studies of memory-related genetic pathways.These cohorts were described as follows.

Sydney Memory and Ageing Study (Sydney MAS)
The Sydney MAS was initiated in 2005 to examine the clinical characteristics and prevalence of mild cognitive impairment and related syndromes, and to determine the rate of change in cognitive function over time 59 .Participants were invited to take a neuropsychological test battery, including among other tests the RAVLT 36 , and Story A of the Wechsler Memory Scale, 3 rd edition, (WMS-III) 60 .DNA was genotyped at the Ramaciotti Centre, UNSW, Australia.Among the 925 participants with available genome-wide data, after exclusion of 37 participants with a history of stroke, 880 individuals were available for performing a GWAS of delayed word list recall (RAVLT) and 887 for delayed paragraph recall (WMS-III).

Women's Genome Health Study (WGHS)
The WGHS is a large cohort for genome-wide genetic analysis of a wide range of clinical phenotypes among >25,000 women, 45 years or older at baseline and with ongoing follow-up observation, now for approximately 18 years 61 .The population is derived from participants in the Women's Health Study who provided a blood sample at baseline.By design, participants included in the WGHS were free from dementia and stroke at baseline.Women above 70 years of age were contacted for a baseline telephone cognitive assessment, including a measure of delayed paragraph recall using the East Boston Memory Test, and the Telephone Interview of Cognitive Status 10-item word list.Genome-wide genotyping in the WGHS was performed using the Illumina (San Diego, CA) HumanHap 300 Duo "+" platform including a total 339,596 SNPs passing quality control filters among 23,294 participants with verified European ancestry.Among participants with available memory tests and genome-wide genotypes, 3,542 individuals of verified European ancestry were available for genetic analysis of delayed word list recall (TICS) and of delayed paragraph recall.

Nurses' Health Study (NHS)
The NHS began in 1976, when 121,700 female registered nurse women, aged 30 to 55 years, living in 11 USA states completed a mailed questionnaire on lifestyle and health.Every 2 years, follow-up questionnaires have been mailed to participants to update their information, and >90% follow-up of the total possible persontime has been maintained 62 .For the study of cognitive function, participants aged 70 years and older free of diagnosed stroke were selected (dementia diagnosis was not ascertained).From 1995 to 2001, 21,085 eligible women were contacted for a baseline telephone cognitive assessment, including a measure of delayed paragraph recall using the East Boston Memory Test, and the Telephone Interview of Cognitive Status 10 -item word list; 19,415 (92%) completed the interview.The study was approved by the Institutional Review Board of the Brigham and Women's Hospital 62 .Genotyping was conducted at the Broad Institute using a nested case-control design.Among participants from 6 independent case-control studies in the NHS cohort with available memory tests and genome-wide genotypes, 2,066 / 1,773 individuals were available for a GWAS of delayed paragraph / word list recall (NHS-CGEM): 756 / 655 from the diabetes casecontrol dataset (NHS-T2D), 358 / 307 from the coronary heart disease case-control dataset, 553 / 450 from the breast cancer case-control dataset (NHS-CHD), 179 / 166 from the glaucoma case-control dataset (NHS-GLAU), 80 / 76 from the kidney stone case-control dataset (NHS-KS), and 141 / 119 from the endometrial cancer case-control dataset (NHS-ENDO).

Samples for the Differential Gene Expression Analyses
We performed a gene-set differential expression (DE) analysis using significant component genes (meta-analysis gene p-value, Gene_p ≤ 1E-06) from verbal declarative memory-associated pathways.Our aim was to examine if these pathway genes collectively had stronger DE by cognitive status in brain tissues compared to an identical number of random genes.Three curated human gene expression studies of cognitive traits were selected from the Gene Expression Omnibus 63 .The first study (ID: GDS4135) tested DE over Braak stages I-II, III-IV, and V-VI of neurofibrillary tangle pathology in astrocytes 64 .The second study (ID: GDS4231) examined DE between HIV-1-associated neurocognitive disorders and uninfected controls in postmortem brain tissue 65 .The third study (ID: GDS4358) investigated the trend of expression changes across uninfected (controls), HIV-1 infected only (HIV-only), HIV-1 infected with substantial neurocognitive impairment (HIV-NCI), and HIV-NCI with HIV encephalitis (HIV-NCI-HIVE) participants in tissue samples from basal ganglia, the frontal cortex, and white matter 66 .For the third study, we performed tests both including (Trend I test) and omitting (Trend II test) the uninfected controls.
Three rodent DE studies were performed using homologues (identified using the NCBI HomoloGene tool 67 ) of the significant memory-associated pathway genes in hippocampal tissue.The first study (ID: GDS2082) investigated DE in 15-month-old house mice with age-related spatial memory deficits versus 2-month-old normal mice 68 .
The second study (ID: GDS2639) compared expression in Norway rats by cognitive impairment status (impaired versus unimpaired cognition) 69 .The third study (ID: GDS520) investigated DE among Norway rates with age-dependent cognitive decline over 4, 14, and 24 months 70 .Gene Id 1 : NCBI gene identifier Gene_p (PAR-dr) 2 : meta-analysis p-value of paragraph delayed recall (PAR-dr) Gene_p (WL-dr) 3 : meta-analysis p-value of word list delayed recall (WL-dr) Significant 4 : both PAR-dr and WL-dr are significant after multiple adjustment (BOTH); only PAR-dr is significant (PAR) and only WL-dr is significant (WL)

GDS2639, GDS520
HLA-DRA RT1-Da RT1 class II, locus Da 294269 GDS2639, GDS520 NOTE: GeneID is the NCBI gene identifier; and GEO_ID: the expression data that has the gene measured.

Figure S3 .
Figure S3.Forest plots of significant pathway enrichment effects and p-values from discovery cohorts (Approach 1) Pathway: KEGG type 1 diabetes N: the number of effective genes in the pathway; M: the number of significant genes; and Padj: permutationadjusted pathway p-value at an individual study.

Table S6 . Significant component genes in the six memory-associated pathways
This table shows the characteristics of the significant component genes from the six memoryassociated pathways.GeneID is the NCBI gene identifier; Gene is the gene name or symbol; Class is the class of the specific MHC gene; chr is the chromosome; start(bp) is the basepair position corresponding to the